Multichannel analysis and monitoring of multiple gene expressions by using bioluminescent reporters

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By transfecting reporter genes which encode luciferase emitting light with different wave lengths, we monitored expression of multiple genes in single cells. The method was applied for two dimensional and time-course analyzes to visualize the interactions of molecules within living cells.(1) Cloning of novel bioluminescence reporter genes and construction of multi-color reporter vector : Two luciferases of the railroad-worm emit red and green lights by catalyzing the same substrate. The dual reporter vector was constructed by inserting the cDNAs, one under the vasopressin and the other under CMV promoter. Although the light emission could be detected, the luminescence in the mammalian cell was weak. Therefore, dual reporter with higher intensity was constructed using vargula hilgendorfii luciferase and firefly luciferase. Promoter sequence of growth hormone (GH) gene was connected to the upstream of vargula luciferase cDNA and the reporter vector was transfected to the GH3 cell. The st … More able colony of GH3 cells was further transfected firefly luciferase. Luminescence from vargula and firefly luciferases was separately monitored from GH3 cells continuously.(2) Construction of multi-color monitoring system : We constructed the detection system consisting of dark box kept 37 ℃ in which a turn table carrying 6 grass-bottom petri dishes rotates on the cooled CCDcamera. A luminocapter counted the photons after separating those with the peak wavelength through liquid crystal filters.(3) Cloning of promoter sequences and continuous monitoring of targeted gene expression in living cells : Transcriptional regulation was analyzed using various length of promoter sequences of mouse Per1 and BMAL1 genes connected with either GFP or firefly luciferase cDNA. We identify the regulatory region in the promoter and constructed dual color reporter to monitor Per1 and BMAL1 expressions. We are constructing the transgenic mice line to study the molecular clock mechanisms monitoring the expression of the two clock genes. Less